Bacteria have been developed for use as vaccines that deliver heterologous antigens. These bacteria have been modified to contain nucleic acid sequences encoding a protein or antigen of interest. However, injection of native virulent infectious bacteria is potentially deleterious to the recipient organism. Therefore, virulence of bacteria has to be attenuated before their use in immunotherapy.
Due to the endowed effective ability to deliver antigen to antigen presenting cells in vivo, type III secretion system (T3SS or TTSS) based attenuated bacterial vectors attracted more and more attention for their potential use, in particular for cancer vaccine development. In the last decades, the use of Gram-negative bacteria, such as Salmonella, Shigella, Yersinia and Pseudomonas which use their powerful secretion machinery—type III secretion system to deliver bacterial effectors to the membrane or into the host cell cytoplasm, has attracted more and more attention for their potential use for cancer vaccine development (Epaulard et al., 2006). Until now, T3SS based bacteria has been proved as a carrier for cancer vaccines which provide the protection against several tumor models like glioma, prostate cancer, breast cancer and fibrosarcoma in mice.
Beside their high efficiency, the safety of bacterial vectors are very important for clinical application. In our previous work described in the article from Epaulard et al., published in 2008, two attenuated strains of Pseudomonas were described:                one attenuated strain “CHA-OST”, in which the exoS exoT genes encoding two major T3S toxin exoenzymes—ExoS and ExoT—have been deleted, demonstrated efficient antigen delivery ability and tumor protection performance;        a more attenuated strain “CHA-OAL”, in which beside exoS and exoT genes, the aroA and IasI genes have also been deleted. The aroA gene encodes the 3-phosphoshikimate 1-carboxyvinyltransferase which is a key enzyme in aromatic amino acid synthesis and the IasI gene encodes the enzyme which produces quorum sensing (QS) homoserine lactones 3-oxo-C12-HSL.        
This second strain CHA-OAL has a greatly reduced toxicity while keeping a good efficiency at high doses. This maximally attenuated strain may represent the best compromise between virulence attenuation and efficiency so that it was endowed the potential for clinical applications.
The patent EP 1 692 162 describes an expression vector of chimeric proteins, comprising the functional part of the proteins ExoS or ExoT from Pseudomonas aeruginosa, and an antigen of interest. The chimeric protein can be expressed by a strain transformed with said vector, and be “injected” into the cytoplasm of an antigen-presenting cell via the TTSS system. Preferentially, the transformed strain is a CHA-OST strain wherein the genes exoS and exoT have been deleted.
Recently, a new concept of vaccines that are “killed but metabolically active” (KBMA) have been described. They retain the immunological properties of live organisms but have a safety profile closer to that of killed organisms. Initially, the KBMA vaccine strategy was demonstrated by Brockstedt et al. with Listeria monocytogenes bacteria (Brockstedt et al., 2005). The deletion of two uvr genes (A and B) coding for exonucleotidase A and B subunit renders bacteria sensitive to psoralen-induced crosslink by exposure to long wavelength UVA light (Wollowitz, 2001). However, it is unclear whether this photochemical treatment could be applied to Pseudomonas bacteria and whether the type III secretion system would work after this treatment.
For clinical purpose, it is also important to join good manufacturing practices for vaccine production. In particular, bacterial vectors should be produced in chemically defined medium with constant growth performance to ensure the quality of the product.
Indeed, one of the main technical problem met with the culture of attenuated Pseudomonas strains is their poor growth rate in standard chemically defined growth media. In recent studies, one chemically defined medium—glucose minimal (M9) medium has been applied in different bacterial species culture, such as E. coli, Salmonella Typhimurium, Pseudomonas putida, for the investigations of genes expression, protein expression and bacterial communities. This medium was previously developed by DeBell R M and had been proved to be ideal for Exotoxin A production by P. aeruginosa (DeBell, 1979).
There is a need in the art for an optimized chemically defined medium, allowing a high and constant growth rate for Pseudomonas attenuated strains. Preferentially, these strains are transformed with a vector of expression such as described in patent EP 1 692 162.